The long term objectives of the project are characterization of the enzymatic activities and gene products involved in synthesis of the peptidoglycan cell wall of bacterial endospores and examination of the role of each type of peptidoglycan structural modification in determining endospore resistance properties. The specific aims are 1) to determine the structure of the endospore peptidoglycan in the first stages of its synthesis and to track the structure to its final form in the dormant spore, 2) to identify changes in synthesis of this structure produced by mutation or loss of each penicillin-binding protein, autolysin, and sporulation-associated gene product, and 3) to purify and characterize in vitro the enzymatic activities involved in peptidoglycan side chain cleavage and muramic lactarn production. Knowledge of principles of endospore resistance properties, dormancy, and longevity may contribute to better decontamination methods and to methods for storage and transport of drugs and vaccines. Peptidoglycan synthesis in general is an attractive target for antibiotic action, further studies of this process will contribute to methods for identification of new antibiotics. Peptidoglycan is purified from sporulating cultures of Bacillus subtilis by chemical and enzymatic treatments, digested with muramidase, and analyzed using high-pressure liquid chromatography (HPLC) using methods previously developed for analysis of dormant spore peptidoglycan. Novel muropeptides are identified using amino acid analysis and mass spectrometry. Appearance and loss of peptide side chain alanine and glycine residues, muramic lactarn production, and changes in peptide cross-linking are quantified throughout the sporulation process. The analysis will be repeated using strains with altered or absent penicillin-binding proteins, autolysins, and sporulation-associated gene products. The cwlD and other gene products found to be associated with muramic lactam production will be assayed in vitro using extracts of sporulating cells or following over-expression in B. subtilis or Escherichia coli. Immature peptidoglycan samples from mutant strains and purified muropeptides will serve as substrates in these assays. Reaction progress will be monitored using the HPLC method and by development of a more rapid capillary electrophoresis method.